Ryanodine receptor inhibitors for treatment of T-cell mediated disorders

ABSTRACT

This invention relates to methods of treating T-cell mediated diseases or disorders in human or animal subjects, such as autoimmune diseases and chronic graft versus host disease in humans and animals. In particular, the methods comprise administering to the human or animal patient a pharmaceutical composition comprising a ryanodine receptor inhibitor.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.12/727,865, filed Mar. 19, 2010, which is a Continuation-in-Part of U.S.application Ser. No. 12/714,198, filed Feb. 26, 2010, which claims thebenefit under 35 U.S.C. §1.119(e) of U.S. provisional Application No.61/208,861, filed Feb. 26, 2009, the contents of which are incorporatedby reference in their entirety.

BACKGROUND OF THE INVENTION

T lymphocytes play a pivotal role in directing the immune responsesagainst foreign pathogens, damaged or transformed cells. However, whenderegulated, T cells may mediate a number of pathological diseases suchas autoimmune diseases (AD), including type-1 diabetes, rheumatoidarthritis, multiple sclerosis, etc., and may significantly affect thedevelopment and progression of inflammatory diseases such ascardiovascular disease and stroke. (Sospedra, M., and Martin, R. (2005)Annu. Rev. Immunol. 23(7): 683-747; Menge, T., Weber, M. S., Hemmer, B.,Kieseier, B. C., von Budingen, H. C., Warnke, C., Zamvil, S. S., Boster,A., Khan, O., Hartung, H. P., and Stuve, O. (2008) Drugs 68(17):2445-68; Cope A. P., Schulze-Koops H., and Aringer M. (2007) Clin ExpRheumatol. 25(5 Suppl 46):S4-11; Yoon J W, and Jun H S. (2005) Am JTher. 12(6):580-91; Skapenko A., Leipe J., Lipsky P. E., andSchulze-Koops H. (2005) Arthritis Res. Ther. 7(Suppl 2):S4-14). Theimmune system is also responsible for the destruction of organtransplants, the so called graft versus host disease (GVHD)(Jacobsohn D.A., and Vogelsang G. B. (2007) Orphanet J. Rare Dis. 4:2:35; Ferrara J.L., Cooke K. R., and Teshima T. (2003) Int. J. Hematol. 78(3):181-7).Currently available therapies for these T cell-mediated diseases arelargely unsatisfactory primarily because all the available drugs areeither only partially effective in reducing exacerbation and slowingdown the disease progression, or produce severe side effects.

Naïve or memory T cells are quiescent cells circulating in the body andare commonly referred to as resting T cells. Resting T cells express Tcell receptors (TCR) that recognize a specific antigen on anantigen-presenting cell. In order to participate in immune response,resting T cells undergo process of activation triggered by TCRcrosslinking with an antigen (Favero, J. and Lafont, V (1998) Biochem.Pharmacol. 56(12): 1539-47; Killeen, N., Irving, B. A., Pippig, S., andZingler, K. (1998) Curr. Opin. Immunol. 10(3): 360-7). During theactivation phase, T cells start to express specific surface receptorsand interleukins, and begin to proliferate with many rounds ofdivisions. In the presence of the repertoire of specific cytokines,activated T cells differentiate into effector T cells that participatein normal immune responses, such as responses directed against foreignpathogens (Swain, S. L. (1999) Curr. Opin. Immunol. 11(2): 180-5). Whenimmune system function is impaired, activated T cells may direct immuneresponses against normal tissues, which causes AD, or against organtransplant, which causes GVHD. Auto-reactive activated T cellsinfiltrate sites of inflammation in patients with AD and are thought todrive the processes of tissue damage via secretion of pro-inflammatorycytokines, such as INF-γ, IL-17, IL-23, and TNF-α.

The process of T cell activation is tightly regulated. Calcium ions(Ca²⁺) play a critical role in T cell activation. In T lymphocytes,sustained elevation in intracellular calcium concentration ([Ca²⁺]_(i))induces a transcriptional program responsible for T cell clonalexpansion and secretion of effector cytokines necessary for coordinationof the immune response (Negulescu, P. A., Shastri, N., and Cahalan, M.D. (1994) Proceedings of the National Academy of Sciences of the UnitedStates of America 91(7), 2873-2877; Dolmetsch, R. E., Lewis, R. S.,Goodnow, C. C., and Healy, J. I. (1997) Nature 386(6627), 855-858;Dolmetsch, R. E., Xu, K., and Lewis, R. S. (1998) Nature 392(6679),933-936). Conversely, diminished [Ca²⁺]_(i) signaling results inimpaired T cell activation and, consequently, development of severeimmunodeficiency (Gwack, Y., Feske, S., Srikanth, S., Hogan, P. G., andRao, A. (2007) Cell Calcium 42(2):145-56; Feske, S. (2007) Nat RevImmunol 7(9), 690-702).

TCR stimulation, by engagement with foreign antigen, for example, causesa biphasic increase in [Ca²⁺]_(i). The first increase results from Ca²⁺release from intracellular stores. This is a consequence of inositol1,4,5-trisphosphate (IP₃) formation. IP₃ binding by the IP₃ receptor(IP₃R) evokes Ca²⁺ release, predominantly from the endoplasmicrecticulum. This first phase is followed by activation of theplasmalemmal store-operated Ca²⁺ (SOC) channels, which allows for thestore-operated Ca²⁺ influx (SOCE) across the plasma membrane (Lewis, R.S. (2001) Annual Review of Immunology 19, 497-521; Putney, J. W., Jr.(1986) Cell Calcium 7(1), 1-12; Putney, J. W., Jr., and McKay, R. R.(1999) Bioessays 21(1), 38-46).

Recent studies have identified members of the ryanodine receptor (RyR)family as contributing to the IP₃-independent component of Ca²⁺signaling in some types of immune cells. RyR antagonists ruthenium redand dantrolene inhibit murine T cell proliferation and IL-2 productionfollowing TCR stimulation, whereas diminished expression of the type 3RyR reduces TCR-dependent [Ca²⁺]i signaling in Jurkat T cells, a humanleukemia cell line derived from a single person (Dupuis, G., and Bastin,B. (1988) J Leukoc Biol 43(3), 238-247; Guse, A. H., da Silva, C. P.,Berg, I., Skapenko, A. L., Weber, K., Heyer, P., Hohenegger, M., Ashamu,G. A., Schulze-Koops, H., Potter, B. V., and Mayr, G. W. (1999) Nature398(6722), 70-73; Schwarzmann, N., Kunerth, S., Weber, K., Mayr, G. W.,and Guse, A. H. (2002) J Biol Chem 277(52), 50636-50642).

In Jurkat T cells TCR activation stimulates production of cyclicadenosine 5′-diphosphate-ribose (cADPR) and/or nicotinic acid adeninedinucleotide phosphate (NAADP), both of which are agonists of the RyRfamily (Guse, A. H., da Silva, C. P., Berg, I., Skapenko, A. L., Weber,K., Heyer, P., Hohenegger, M., Ashamu, G. A., Schulze-Koops, H., Potter,B. V., and Mayr, G. W. (1999) Nature 398(6722), 70-73; Gasser, A.,Glassmeier, G., Fliegert, R., Langhorst, M. F., Meinke, S., Hein, D.,Kruger, S., Weber, K., Heiner, I., Oppenheimer, N., Schwarz, J. R., andGuse, A. H. (2006) J. Biol. Chem. 281(5), 2489-2496). Decreased cADPRlevels also reduce the TCR-dependent [Ca²⁺]_(i) signaling in Jurkat Tcells (Guse, A. H., da Silva, C. P., Berg, I., Skapenko, A. L., Weber,K., Heyer, P., Hohenegger, M., Ashamu, G. A., Schulze-Koops, H., Potter,B. V., and Mayr, G. W. (1999) Nature 398(6722), 70-73; Schwarzmann, N.,Kunerth, S., Weber, K., Mayr, G. W., and Guse, A. H. (2002) J. Biol.Chem. 277(52), 50636-50642). Mechanisms of activation, and the role ofRyR in regulation of [Ca²⁺]_(i) signaling and Ca²⁺-dependent functionsin normal human T cells have not been previously elucidated. Recentstudies described in this invention revealed for the first time that RyRare highly expressed in activated human T cells, where they areactivated by SOCE and play essential role in controlling Ca²⁺ signalingand Ca²⁺-dependent functions of activated T lymphocytes.

There are three known isoforms of RyR: type 1 (RyR1), type 2 (RyR2), andtype 3 (RyR3). The RyR1 and RyR2 were originally found in thesarcoplasmic reticulum of skeletal and cardiac muscles, respectively.Ca²⁺ release from the sarcoplasmic reticulum through these receptorsplays a central role in regulating the contraction of skeletal andcardiac muscle fibers. The RyR3 has been detected in specific regions ofthe brain, nonmuscle tissues, and skeletal muscle as well.

BRIEF SUMMARY OF THE INVENTION

The present invention is based on the surprising finding that RyR aregenetically and functionally up-regulated in activated human T cells,and that inhibitors of type 1 and type 3 RyR suppress intracellular Ca²⁺signaling and Ca²⁺-dependent functions of activated human T lymphocytes.Therefore, inhibitors of RyR1 and/or RyR3 can be used asimmunosuppressants and/or for the treatment of T cell-mediated humandisorders, such as autoimmune disorders (multiple sclerosis, rheumatoidarthritis), graft versus host diseases (e.g., graft rejection), andinflammation.

The present invention provides methods of treating or preventing aT-cell mediated disorders in humans and animals. The methods compriseadministering to the human or animal patient, in the amount and formthat is effective to treat or prevent the disease or disorder, apharmaceutical composition comprising a ryanodine receptor inhibitor anda pharmaceutically acceptable carrier, wherein the patient is notsuffering from muscular skeletal dysfunction.

The T-cell mediated disorder can be inflammation, an autoimmune disease,or graft versus host disease. In some embodiments, the autoimmunedisease is multiple sclerosis.

In some embodiments, the patient is diagnosed with relapsing-remittingor primary progressive clinically definite multiple sclerosis, orpresents a first clinical demyelinating event and multiple sclerosis ishighly suspected.

The RyR inhibitor can be any compound or agent that inhibits calciumrelease from the ryanodine receptor(s). In some embodiments, the RyRinhibitor is specific for type 1 RyR. In some embodiments, the RyRinhibitor is specific for type 3 RyR. In some embodiments, the RyRinhibitor is specific for type 1 and type 3 RyR. In some embodiments,the RyR inhibitor has an affinity for type 1 and/or type 3 RyR at least10 fold higher than its affinity for type 2 RyR. The RyR inhibitor ofthe present invention can be administered orally, parenterally, and/ortopically.

In some embodiments, the compound is dantrolene, a dantrolene analogue,or a pharmaceutically acceptable salt thereof. An exemplary analogue isazumolene. In some embodiments, the pharmaceutically acceptable salt isa sodium salt. In some embodiments, the pharmaceutically acceptable saltis a quaternary ammonium salt. In some embodiments, the compound is anatural or synthetic ryanoid. In some embodiments, the compound isryanodine. In other embodiments, an inhibitory nucleic acid molecule,such as an siRNA, can be used to inhibit expression of the ryanodinereceptor, e.g., specifically RyR1 and/or RyR3.

The methods are applicable to both human therapy and veterinaryapplications. In the preferred embodiment the patient is a mammal,preferably a primate, and in the most preferred embodiment the patientis human. In some embodiments, the patient is a non-human.

DEFINITIONS

A ryanodine receptor inhibitor is a compound or agent that inhibits orreduces calcium efflux from the ryanodine receptor. The agent can be asmall molecule antagonist such as dantrolene(1-[[[5-(4-nitrophenyl)-2-furanyl]methylene]amino]-2,4-imidazolidinedione) or a water-soluble analogue thereof, such as azumolene(1-[[[5-(4-bromophenyl)-2-oxazolyl]methylene]amino]-2,4-imidazolidinedione) whose biological activity is conferred to inhibition ofRyR1 and/or RyR3, but not RyR2. Other dantrolene analogues useful forthe present invention include1-{[5-(4-Methoxyphenyl)furfurylidene]amino}imidazolidine-2,4-dione,1-{[5-(3-Methoxyphenyl)furfurylidene]amino}imidazolidine-2,4-dione,1-{[5-(2-Methoxyphenyl)furfurylidene]amino}imidazolidine-2,4-dione,1-[(5-Phenylfurfurylidene)amino]imidazolidine-2,4-dione,1-{[5-(4-Methylphenyl)furfurylidene]amino}imidazolidine-2,4-dione,1-{[5-(4-Nitrophenyl)furfurylidene]amino}imidazolidine-2,4-dione,1-{[5-(3-Nitrophenyl)furfurylidene]amino}imidazolidine-2,4-dione,1-{[5-(2-Nitrophenyl)furfurylidene]amino}imidazolidine-2,4-dione,1-{[5-(4-Trifluoromethylphenyl)furfurylidene]amino}imidazolidine-2,4-dione,1-{[5-(2,3,4,5,6-Pentafluorophenyl)furfurylidene]amino}imidazolidine-2,4-dione,1-{[5-(4-Fluorophenyl)furfurylidene]amino}imidazolidine-2,4-dione,1-{[5-(4-Phenylphenyl)furfurylidene]amino}imidazolidine-2,4-dione,1-{[5-(2,6-Dinitrophenyl)furfurylidene]amino}imidazolidine-2,4-dione,1-{[5-(1-Naphthyl)furfurylidene]amino}imidazolidine-2,4-dione,1-{[5-(4-Bromophenyl)furfurylidene]amino}imidazolidine-2,4-dione,1-{[5-(4-Hydroxyphenyl)furfurylidene]amino}imidazolidine-2,4-dione,1-{[5-(4-Trifluoromethanesufonylphenyl)furfurylidene]amino}imidazolidine-2,4-dione,1-{[5-(4-Cyanophenyl)furfurylidene]amino}imidazolidine-2,4-dione,1-{[5-(4-Nitrophenyl)-2-thenylidene]amino}imidazolidine-2,4-dione,1-{[5-(2-Nitrophenyl)-2-thenylidene]amino}imidazolidine-2,4-dione(Hosoya T, Aoyama H, Ikemoto T, Kihara Y, Hiramatsu T, Endo M, Suzuki M.(2003), Bioorg Med Chem. 11(5):663-73). Other exemplary small moleculeantagonists include 1) ryanoids (e.g. ryanodine (an ester ofpyrrole-α-carboxylic acid with ryandolol), 9,21-didehydroryanodine(differs from ryanodine by the absence of two H atoms), or other naturalor synthetic biologically active ryanoids; 2) purine derivatives andrelated compounds (e.g. 4,6-dibromo-3-hydroxycarbazole a carbazolederivative with a carbazole skeleton and bromine at C-6); 3)anthraquinones (e.g. doxorubicin); 4) polyamines: inorganic polyamines(e.g. polycationic dye ruthenium red; tetramine palladium and tetramineplatinum), aminoglycoside antibiotics (e.g. neomycin, gentamicin,streptomycin); organic polyamines (e.g. polylysine and polyarginine); 5)FLA365. [2,6-dichloro-4-(dimethylamino)phenyl]-isopropylamine; 6) localanesthetics (e.g. procaine and tetracaine, lidocaine, QX 314); 7)peptides (e.g. imperatoxin-I, helothermine).

Alternatively, the inhibitor can be biological molecule such as aninhibitory nucleic acid, such as antisense nucleic acids, shortinterfering RNA (siRNA), small hairpin RNA (shRNA), microRNA, ribozymesor similar molecules which downregulate a specific type of RyR (e.g.,RyR1 and/or RyR3) gene or protein expression. In some embodiments, theinhibitory nucleic acid is a small interfering RNA that preventstranslation of RyR1 and/or RyR3 mRNAs. In some embodiments, theinhibitory nucleic acid is a small hairpin RNA that prevents translationof RyR1 and/or RyR3 mRNAs.

The term “siRNA” refers to a double stranded RNA molecule which preventstranslation of a target mRNA. Standard techniques are used forintroducing siRNA into cells, including those wherein DNA is used as thetemplate to transcribe RNA. The siRNA comprises a sense nucleic acidsequence and an anti-sense nucleic acid sequence of the polynucleotideof interest. The siRNA is constructed such that a single transcript(double stranded RNA) has both the sense and complementary antisensesequences from the target gene, e.g., a hairpin.

The nucleotide sequence of siRNAs may be designed using an siRNA designcomputer program available from, for example, the Ambion website on theworld wide web. Nucleotide sequences for the siRNA are selected by thecomputer program based on the following protocol:

Selection of siRNA Target Sites:

-   1. Beginning with the AUG start codon of the object transcript, scan    downstream for AA dinucleotide sequences. Record the occurrence of    each AA and the 3′ adjacent 19 nucleotides as potential siRNA target    sites. Tuschl, et al. recommend against designing siRNA to the 5′    and 3′ untranslated regions (UTRs) and regions near the start codon    (within 75 bases) as these may be richer in regulatory protein    binding sites. UTR-binding proteins and/or translation initiation    complexes may interfere with the binding of the siRNA endonuclease    complex.-   2. Compare the potential target sites to the human genome database    and eliminate from consideration any target sequences with    significant homology to other coding sequences. The homology search    can be performed using BLAST, which can be found on the NCBI server    on the world wide web.-   3. Select qualifying target sequences for synthesis. At Ambion,    preferably several target sequences can be selected along the length    of the gene for evaluation.

The inhibitory nucleic acids of the invention will inhibit theexpression of a specific type of RyR and is thereby useful insuppressing its biological activity. In some embodiments, the length ofthe antisense oligonucleotides and siRNAs is less than or equals to 10nucleotides. In some embodiments, the length of the antisenseoligonucleotides and siRNAs is as long as the naturally occurring thetranscript. In some embodiments, the antisense oligonucleotides andsiRNAs have 19-25 nucleotides. In some embodiments, the antisenseoligonucleotides and siRNAs are less than 75, 50, 25 nucleotides inlength.

A “patient” for the purposes of the present invention includes bothhumans and other animals, particularly mammals.

Muscular skeletal dysfunction, as used herein, refers to conditionsassociated with weakness and/or wasting of skeletal muscles, whichincludes symptoms such as lower back pain, joint injuries and variousrepetitive strain injuries, including upper limb disorder(s) affectingthe arms, neck and shoulders.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Genetic and functional expression of human RyR1, RyR2, and RyR3in human peripheral blood mononuclear cell-derived resting and activatedT cells. (A), Representative quantitative TaqMan PCR amplification plotsfor hRyR1 amplification; the fluorescence intensities are plotted asfunctions of a cycle number. Each TaqMan PCR analysis was performed induplicates. C_(t) value, a cycle number in which fluorescence reaches athreshold level (red line), was used for quantification. (B),Quantification of data presented in (A) showing relative expression ofhRyR1, hRyR2, and hRyR3 in resting (R, open bars) and activated (A, greybars) T lymphocytes from nine healthy human donors determined using RealTime TaqMan PCR. B2M housekeeping gene expression was used fornormalization. Final quantization was done using the comparative C_(t)method (User Bulletin #2, Applied Biosys.) and is reported astranscription relative to hRyR1, hRyR2, and hRyR3 cDNA levels in restingT cells from a randomly selected donor. In brief, the housekeeping gene,B2M, was used to normalize the C_(t) values of the target genes(ΔC_(t)=C_(t)(hRyR)−C_(t)(B2M)). The ΔC_(t) was calibrated against theΔC_(t) of the target gene (hRyR1, hRyR2, or hRyR3) in resting T cellsfrom a randomly selected donor. The relative linear amount of targetmolecules relative to the calibrator, was calculated by 2^(−ΔΔCt). (*)indicates that differences between means are significant (p<0.05;independent Student's t test; n=9). n—number of experiments; errorbars=S.E. of the means. (C), Time courses of changes in cytosolic Ca²⁺concentration ([Ca^(2+]) _(i)) in response to RyR agonist caffeine (20mM) recorded from resting (top panel), or activated human T cells (lowerpanel), in normal Tyrode extracellular solution. To confirm thatcaffeine evokes Ca²⁺ release from the RyR, activated T cells werepre-incubated with 400 μM ryanodine (Ry, blue trace), which at thisconcentration blocks all types of RyR, or vehicle alone (Control, blacktrace) for 30 min prior to the application of caffeine. Each tracerepresents an average of 20 traces recorded from the individual cells.

FIG. 2. RyR inhibitor ryanodine (Ry) reduces global [Ca²⁺]_(i) elevationduring Ca²⁺ readdition and facilitates store refilling in Jurkat Tlymphocytes. (A), Average [Ca²⁺]_(i) time courses recorded fromuntreated cells (black traces, control) or cells pre-incubated with Ry(green trace). Extracellular Ca²⁺ was omitted prior to the recordings.Cyclopiazonic acid (CPA, 30 μM) and 2 mM Ca²⁺-containing extracellularsolution were applied as indicated. Note that, on average, cellspre-incubated with Ry display reduced [Ca²⁺]_(i) transient followingCa²⁺ readdition but enhanced [Ca²⁺]_(i) transients induced by the secondCPA application. Each trace is an average of 20 traces recorded from theindividual cells in a typical experiment. Areas under the [Ca²⁺]_(i)traces were integrated to determine amount of global [Ca²⁺]_(i)elevation during Ca²⁺ readdition ([Ca²⁺]_(SOCE)) and amount of Ca²⁺released from the store by application of CPA prior (CPA₁) and after(CPA₂) Ca²⁺ readdition. Values of [Ca²⁺]_(i) prior to the CPA orextracellular Ca²⁺ readdition were subtracted before integration. Ratioof integrated responses to CPA (CPA₂/CPA₁) was taken as a measure ofstore refilling following Ca²⁺ readdition. (B, C), Summarized data fromexperiments performed as shown in (A). (B), Average [Ca²⁺]_(SOCE) inuntreated cells (open bars; control) and in cells preincubated with Ry(grey bars). (C) Average CPA₂/CPA₁ ratio in untreated cells (open bars;control) and in cells pre-incubated with Ry (grey bars). Stars indicatethat differences between means are statistically significant (p<0.01,independent Student's t test, n=6; n—number of experiments). (D), Timecourses of changes in cytosolic free Ca²⁺ concentration ([Ca²⁺]_(i))showing the absence of acute effects of Ry (400 μM, green trace) appliedin nominally Ca²⁺-free extracellular solution on. Vehicle (DMSO) alonewas applied instead of Ry in control experiments (black trace).Extracellular Ca²⁺ was omitted and ryanodine (Ry, RyR blocker) orcyclopiazonic acid (CPA), a blocker of sarco-endoplasmic reticulum Ca²⁺ATPase, were applied as indicated. Each trace is an average of 20 tracesrecorded from individual cells in a typical experiment (n=5, n—number ofexperiments). (E), Time courses of Mn²⁺ quench of fura-2 fluorescence(F_(i)) in untreated cells (black traces, control) and cellspreincubated for 30 min with 400 μM Ry (green traces). Baseline timecourses of Mn²⁺ quench of fura-2 fluorescence were recorded in theabsence of CPA (upper traces). To record Mn²⁺ influx via SOC channels,30 μM CPA was applied 10 min prior to Mn²⁺ to deplete the store (lowertraces). Each trace is an average of 20 traces recorded from individualcells in a typical experiment (n=5).

FIG. 3. Ryanodine (Ry) downregulates Ca²⁺ responses to T cell receptor(TCR) agonist phytohaemagglutinin P (PHA). (A, B), Average [Ca²⁺]_(i)responses to 20 μM PHA applied as indicated in Ca²⁺-free externalsolution in cells preincubated with 400 μM Ry (B, blue trace) orvehicles alone (black traces, control). 30 μM CPA or 0.5 mMCa²⁺-containing solution were applied following PHA as indicated. Eachtrace represents an average of 20 traces recorded from individual cells.(C-E), Summarized data from experiments performed as shown in (A and B).(C), Average integrated [Ca²⁺]_(i) transients evoked by PHA([Ca²⁺]_(PHA)) in cells preincubated with Ry (grey bars), or vehiclesalone (open bars; control). (D), Average integrated [Ca²⁺]_(i)transients evoked by CPA ([Ca²⁺]_(CPA)) applied following PHA in cellspre-incubated with Ry (grey bars), or vehicles alone (open bars;control). Brackets in (A and B) indicate the areas that werebackground-subtracted and integrated to obtain [Ca²⁺]_(PHA) and[Ca²⁺]_(CPA) values. (E), Average baseline-subtracted peak values of[Ca²⁺]_(i) transients, shown in (B), evoked by 0.5 mM Ca²⁺ readdition incells preincubated with Ry (grey bars), or vehicles alone (open bars;control). (*) indicates that differences between means are statisticallysignificant at p<0.01 (independent t test). n—number of experiments.

FIG. 4. RyR inhibitors ryanodine (Ry) and dantrolene (Da) suppressglobal [Ca²⁺]_(i) elevation in activated but not resting human Tlymphocytes. (A), Average [Ca²⁺]_(i) time courses recorded from restingT cells pre-incubated for 30 minutes prior to the experiment with Ry(500 μM, blue trace), or Da (30 μM, red trace) or vehicles alone (MetOHor EtOH; control, black trace). Each trace is an average of 18-20 tracesrecorded from individual cells in a typical experiment. Representativetraces from one of seven experiments performed on resting T cells fromfive different donors. (B), Average [Ca²⁺]_(i) time courses recordedfrom activated T cells preincubated for 30 minutes prior to theexperiment with vehicles alone (MetOH or EtOH; control, black trace). orRy (500 μM, blue trace) or Da (30 μM, red trace). CPA (30 μM), and 2 mMCa²⁺ were applied as indicated. To obtain activated T cell population,resting T cells where stimulated in vitro with immobilized anti-CD3antibodies (Ab) and soluble anti-CD28 Ab (anti-CD3/CD28 Ab) for 72-96 h.Each trace is an average of 18-20 traces recorded from the individualcells in a typical experiment. Representative traces from one of sevenexperiments performed on activated T cells from five different donors.(C), Summarized effects of RyR blockers Ry (500 μM) and Da (30 μM) onglobal [Ca²⁺]_(i) elevation following activation of SOCE ([Ca²⁺]_(SOCE))in activated human T cells. [Ca²⁺]_(SOCE) was determined by integrationof the area under the [Ca²⁺]_(i) trace during readdition of extracellular Ca²⁺ following CPA wash out as shown in (A and B). Values of[Ca²⁺]_(i) prior to the extracellular Ca²⁺ readdition were subtractedbefore integration. (*) indicate that differences between means arestatistically significant (p<0.01, independent Student's t test; n=7).n=number of experiments; error bars=S.E. of the means.

FIG. 5. RyR inhibitors ryanodine (Ry) or dantrolene (Da) inhibit Jurkatand normal human activated T cell proliferation and IL-2 production.(A), The fluorescence profile of CFSE-loaded Jurkat T cells (top panels)and human T cells (lower panels) incubated for 72 hours in the presenceof 400 μM Ry (left panels; shaded histograms) or 30 μM Da (right panels;shaded histograms), or vehicles alone (control, open histograms in allpanels). The dashed lines show the fluorescence profiles of CFSE-loadedcells incubated for 12 hours in FBS-deprived media (undivided cellpopulation at time 0). The brackets and numbers above the bracketsindicate an estimated fraction of undivided cells in control cellpopulations and cell populations incubated with Ry or Da. Representativedata from five experiments performed with each blocker. (B), IL-2content in Jurkat T cell culture supernatants. Cells were incubated for24 hours in cell culture media supplemented with 20 μM PHA alone or incombination with Ry (400 μM) or Da (30 μM), as indicated with (+); orvehicles alone, as indicated with (−). The Ry or Da were applied 30 minprior to the PHA. (*) indicates that differences between means arestatistically significant at p<0.01 (independent Student's t test, n=6for each type of experiments).

FIG. 6. Simplified hypothetical scheme of Ca²⁺ dynamics in T cells uponTCR activation. (A), Diagram of intracellular Ca²⁺ dynamics in T cellsfollowing TCR activation. (B), proposed mechanism of immunosuppressiveaction of RyR inhibitors (e.g. ryanodine, dantrolene, azumolene, RyRsiRNA, etc) inhibition of RyR reduces intracellular Ca²⁺ elevationfollowing TCR engagement, which in turn leads to incomplete genetranscription program and inhibition or modification of T cellactivation or differentiation. Abbreviations: TCR—T cell receptor,A—antigen, IP₃R—inositol 1,4,5-trisphosphate receptor, RyR—ryanodinereceptor; SOC—store-operated Ca²⁺ channel, ER—endoplasmic reticulum.SERCA and PMCA are omitted for simplicity.

DETAILED DESCRIPTION

The immune system is a collection of biological processes within anorganism that protects against disease by identifying and killingpathogens and tumor cells. It detects a wide variety of agents, fromviruses to parasitic worms, and needs to distinguish these from theorganism's own healthy cells and tissues in order to function properly.

The immune system can cause disease and harm to the organism.Autoimmunity results from a hyperactive immune system attacking normaltissues as if they were foreign organisms. Even when the immune systemis functioning normally, it can have detrimental effects on theorganism. The immune system is responsible for the destruction of organtransplants, the so called graft versus host disease (GVHD).

Autoimmune diseases are characterized by T and B lymphocytes thataberrantly target self-proteins and/or other self-molecules causinginjury and or malfunction of an organ, tissue, or cell-type within thebody (for example, pancreas, brain, thyroid or gastrointestinal tract).Autoimmune diseases include diseases that affect specific tissues aswell as diseases that can affect multiple tissues. The characteristicfeature of tissue-specific autoimmunity is the selective targeting of asingle tissue or individual cell type. Nevertheless, certain autoimmunediseases that target ubiquitous self-proteins can also effect specifictissues.

T-lymphocytes are believed to contribute to the development of a numberof autoimmune disease. Table 1 provides a non-exhaustive list ofautoimmune diseases or disorders caused by T cells.

TABLE 1 Nervous System Multiple sclerosis Myasthenia gravis Autoimmuneneuropathies such as Guillain-Barré Autoimmune uveitis Ophthalmologic:Uveitis Blood: Autoimmune hemolytic anemia Pernicious anemia Autoimmunethrombocytopenia Vascular: Temporal arteritis Anti-phospholipid syndromeVasculitides such as Wegener's granulomatosis Behcet's diseaseAtherosclerosis Skin: Psoriasis Dermatitis herpetiformis Pemphigusvulgaris Vitiligo Pemphigus Vulgaris Mycosis Fungoides Allergic ContactDermatitis, Atopic Dermatitis Lichen Planus PLEVA (Pityriasislichenoides et varioliforms acuta), Gastrointestinal Tract Crohn'sDisease Ulcerative colitis Primary biliary cirrhosis Autoimmunehepatitis Endocrine: Type 1 diabetes mellitus Addison's Disease Grave'sDisease Hashimoto's thyroiditis Autoimmune oophoritis and orchitisAutoimmune Thyroiditis Multiple Organs and/or Musculoskeletal System:Rheumatoid arthritis Systemic lupus erythematosus SclerodermaPolymyositis Dermatomyositis Spondyloarthropathies such as ankylosingspondylitis Sjogren's Syndrome

A Prototypic Autoimmune Disorder: Multiple Sclerosis

Multiple sclerosis (MS; also known as disseminated sclerosis orencephalomyelitis disseminata) is an autoimmune disease affecting thenervous system. In MS the immune system attacks and damages myelin, aprotein which surrounds nerve cells. This damage disrupts the ability ofnerve cells in the brain and spinal cord to communicate with each other.

MS is the most common disabling neurologic disease in people ages 18 to60. Disease onset usually occurs in young adults, and it is more commonin females. What triggers MS remains unknown.

A. MS Symptomology

Initial MS symptoms usually present themselves as episodic acute periodsof worsening (relapsing-remitting from the disease) and can varydepending on which part of the nervous system has been affected, anddegree of neuron distraction (Compston, A, et al. McAlpine's MultipleSclerosis. 3d ed. London: Churchill Livingston; 1998; Clanet, Michel G.;Brassat, David Curr Opin Neurol 2000 June; 13(3):263-70; MS SocietySymptom Management Survey. London: MS Society; 1997; Noseworthy J. H.,Lucchinetti C., Rodriguez M., et al. 2000. Multiple sclerosis. N Engl JMed, 343: pp 938-952; Simon, J H, 2006, Update on Multiple Sclerosis,Radiol Clin N Am, 44: 79-100). At the onset of disease or, at theinitial stage of relapsing remitting period, symptoms can include opticneuritis, nystagmus resulting from internuclear ophthalmoplegia, and anelectrical sensation extending down the back and legs with flexion ofthe neck. Patients may also present with different neurologic symptoms,including diplopia, bladder dysfunction, vertigo, weakness,paresthesias, and sensory loss. As the disease progresses, the symptomstend to worsen and other symptoms such as weakness, stiffness,proprioceptive loss of the lower limbs and locomotor ataxia become moresignificant. The last and most debilitating stages of disease includesymptoms secondary to the loss of major neurological functions:spasticity and spasms, urinary incontinence and tract infections, actionor truncal tremor, paralysis and pressure sores, nystagmus and abnormaleye movements, neurological chronic pain and cognitive impairment.

B. Guidelines for Diagnosis

The most current guidelines for diagnosing clinically definite multiplesclerosis (CDMS) came from the deliberations of the International Panelon the Diagnosis of Multiple Sclerosis and are known as the McDonaldcriteria (McDonald W. I., Compston A., Edan G., et al. 2001. Recommendeddiagnostic criteria for multiple sclerosis: guidelines from theInternational Panel on the Diagnosis of Multiple Sclerosis. Ann Neurol50: pp 121-127; Polman C H, Reingold S C, Edan G, et al., 2005.Diagnostic criteria for multiple sclerosis: 2005 revisions to the“McDonald Criteria.” Ann Neurol; 58:840-846). They use a combination ofclinical symptoms, MRI observations and other objective laboratoryfindings to establish dissemination of disease in time and space typicalfor MS.

The McDonald criteria allow for an earlier diagnosis of CDMS but arecriticized for being too rigorous, leading to the case in which CDMSremains undiagnosed despite growing clinical symptoms (Woo D A et al.(2006) Diagnosis and Management of Multiple Sclerosis: Case Studies.Neurologic Clinics 24(2):199-214). Therefore, other criteria andpredictors for CDMS continue to emerge. The MRI findings at symptomonset were found to be the most important predictor of conversion toCDMS. The late multiphasic disease was found to be associated withdisseminated lesions in combination with the optic nerve at the onset ofdisease, whereas a normal MRI is associated with a lower risk ofconversion to CDMS (Frohman E M, Goodin D S, Calabresi P A, et al., theutility of MRI in Suspected MS: report of the Therapeutics andTechnology Assessment Subcommittee for the American Academy ofNeurology. Neurology. 61:602-611).

The earliest clinical presentation of relapsing-remitting MS is a singleattack of a single symptom, which is termed a clinically isolatedsyndrome (CIS). The CIS is suggestive of demyelination but two or moreattacks disseminated in time are necessary to fulfill the McDonaldcriteria for MS diagnosis.

Although exact conversion rates of all CIS to CDMS are not known, it wasshown that the risk of conversion from optic neuritis to CDMS during 10years following initial episode ranged from 22% to 56% (Beck R. W.,Trobe J. D., Moke P. S., et al. 2003. High- and low-risk profiles forthe development of multiple sclerosis within 10 years after opticneuritis. Arch Ophthalmol., 121: pp 944-949). Furthermore, a review bythe Therapeutics and Technology Assessment Subcommittee of the AmericanAcademy of Neurology, demonstrated hidden disease activity in 50 to 80%of patients at the time of the first clinical presentation.

C. Protocols for Treating Multiple Sclerosis

Patients with MS are usually categorized into the following four groups:

-   -   1. Relapsing-remitting (RR) disease is the most common initial        course of the disease which occurs at onset in 80% of cases and        is characterized by acute attacks followed by remissions between        attacks.    -   2. In 50% to 80% of patients with RR disease, progressive        deterioration with less marked attacks occurs within 10 years of        onset; the disease in these patients is called secondary        progressive phase MS (SP-MS).    -   3. Primary progressive MS (PP-MS) occurs in 10% to 15% of        patients and is characterized by progressive deterioration from        the outset without relapses.    -   4. Approximately 6% of patients have progressive-relapsing MS        (PP-MS) wherein patients experience relapses in parallel with        the steady disease progression.

The treatment of MS has three main components: disease modifyingtreatments, relapse treatments, and symptoms management. The diseasemodifying treatments aim to reduce the risk of relapse number andseverity, the development of new brain or spinal cord lesions, and theprogression of neurologic disability. The disease modifying treatmentsexert their effects through an anti-inflammatory, immunomodulatory, orimmunosuppressive mechanisms. Because of the progressive nature of theMS, the disease modifying treatments are most effective when started atthe onset of the disease.

The patient may benefit from the disease modifying treatments even ifthey do not completely fulfill the McDonald criteria for CDMS. Therapycan be started if MS is highly suspected and other diseases have beenreasonably excluded. For example, two randomized, controlled trialsdemonstrated a treatment benefit in patients with a CIS and MRIabnormalities suggestive that MS patients benefited from treatment withinterferon β (IFNβ)(Jacobs L D, Beck R W, Simon J H, et al. 2000.Intramuscular interferon beta-1a therapy initiated during a firstdemyelinating event in multiple sclerosis. N Engl J Med; 343: 898-904;Comi G, Filippi M, Barkhof F, et al. 2001. Effect of early interferontreatment on conversion to definite multiple sclerosis: a randomizedstudy. Lancet; 357: 1576-1582. These results support the use of thedisease modifying treatments after a first clinical demyelinating eventand indicate that there may be modest beneficial effects of immediatetreatment compared with delayed initiation of treatment.

Organ Transplantation

A common complication in tissue and organ transplantation in humans isimmunological incompatibility between the tissue transplant and thehost. Graft versus host disease (GVHD) causes significant morbidity andmortality in patients receiving transplants containing allogeneichematopoietic cells. It is well established that the greater thematching of the MHC class I and II (HLA-A, HLA-B, and HLA-DR) allelesbetween donor and recipient the better the graft survival and fewercomplications with GVHD. Approximately 50% of patients receiving atransplant from a HLA-matched sibling will develop moderate to severeGVHD, and the incidence is much higher in non-HLA-matched grafts.One-third of patients that develop moderate to severe GVHD will die as aresult. T lymphocytes and other immune cell in the donor graft attackthe recipients' cells that express polypeptides variations in theiramino acid sequences, particularly variations in proteins encoded in themajor histocompatibility complex (MHC) gene complex on chromosome 6 inhumans. GVHD is treated with glucocorticoids, cyclosporine,methotrexate, fludarabine, and OKT3.

Modalities for Treating Immune Cell Mediated Disease

Currently available therapies for these immune cell mediated diseases(e.g., autoimmunity, GVHD) are largely unsatisfactory and typicallyinvolve the use of glucocorticoids (e.g. methylprednisolone,prednisone), non-steroidal anti-inflammatory agents, gold salts,methotrexate, antimalarials, and other immunosuppressants such ascyclosporin and FK-506. The usefulness of these agents is limited oftenby severe side effects such as liver and renal damage. Furthermore, notall patients are responsive to all therapies. For example, it is knownthat 30% of multiple sclerosis patients are non-responsive toβ-interferon (Bertolotto, A and Gilli, F. (2008). Interferon-betaresponders and non-responders. A biological approach. NeurologicalSciences: Official Journal of the Italian Neurological Society and ofthe Italian Society of Clinical Neurophysiology. 29 Suppl. 2: S216).Additionally, for Multiple Sclerosis in particular, the current therapywith interferon-beta and copaxone only benefits about 60% of patients.The appearance of neutralizing antibodies in around 40% of patientstreated with interferon-beta makes interferon-beta treatment lesseffective over time in the responsive patients. As a result, the vastmajority of AD patients continue to accrue progressive disabilitydespite immunomodulatory therapies. Moreover, approximately 50% ofpatients receiving a transplant from a HLA-matched sibling developmoderate to severe GVHD, and the incidence is much higher innon-HLA-matched grafts. One-third of patients that develop moderate tosevere GVHD will die as a result. Finally, the pathogenesis of AD andGVHD is complex and may be heterogeneous across patients or inindividual patients over time. A combination regimen could potentiallytreat multiple distinct pathogenic mechanisms better than any singleagent. Therapies with partial efficacy alone could have additive orsynergistic efficacy in combination and allow for lower doses withdecreased adverse effects.

A ryanodine receptor inhibitor, e.g., a compound or agent that inhibitsor reduces calcium efflux from the ryanodine receptor, can be used inthe methods of the present invention for treating or preventing a T-cellmediated disorder. In some embodiments, the inhibitor inhibits allisotypes of RyR. In some embodiments, the inhibitor is an inhibitorspecific for a subset of RyRs. For example, inhibitors useful for thepresent invention include RyR inhibitors specific for RyR1, RyR3, orboth. In some embodiments, the inhibitor does not inhibit RyR2.

In some embodiments, selective inhibitors of the RyR1 and RyR3, such asdantrolene or azumolene, are less likely to cause such side effects and,thus, may be used alone or in combination with other agents (e.g.,cyclosporin and/or FK506) to treat autoimmune diseases and/or GVHD.Thus, the RyR1 and RyR3 inhibitors disclosed herein may providesubstantial improvements in the treatment of autoimmune diseases and/orGVHD.

Still further in accordance with the present invention, there areprovided methods for causing a desired inhibition of RyR1 and/or RyR3while not causing undesired inhibition of RyR2 (cardiac type) in a humanor animal subject. Such methods generally comprise the RyR blockers thata) selectively inhibit RyR1 and/or RyR3 while b) not causing inhibitionof RyR2.

The “desired inhibition of RyR1 and/or RyR3” can be, for example, anyinhibition of RyR1 and/or RyR3 that causes an intended therapeutic orpreventative effect, such as suppression of function of RyR1 and/or RyR3in T lymphocytes to treat or prevent a T cell-mediated disorder in thehuman or animal patient. The “undesired inhibition of RyR2” can be, forexample, any inhibition of a RyR2 that causes cardiac side effects,untoward effect or any effect other than the desired therapeutic orpreventative effect, such as the inhibition of RyR2 in a way that causesa proarrhythmic effect or increases the potential for cardiac arrhythmiain the human or animal patient.

The agents that inhibit ryanodine receptors can be administered by avariety of methods including, but not limited to parenteral (e.g.,intravenous, intramuscular, intradermal, intraperitoneal, andsubcutaneous routes), topical, oral, local, or transdermaladministration. These methods can be used for prophylactic and/ortherapeutic treatment.

As noted above, inhibitors of the invention can be used to treat T-cellmediated disorders. The compositions for administration will commonlycomprise a inhibitor dissolved in a pharmaceutically acceptable carrier,preferably an aqueous carrier. A variety of aqueous carriers can beused, e.g., buffered saline and the like. These solutions are sterileand generally free of undesirable matter. These compositions may besterilized by conventional, well known sterilization techniques. Thecompositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate water solubility and/orphysiological conditions such as pH adjusting and buffering agents,toxicity adjusting agents and the like, for example, sodium acetate,sodium chloride, potassium chloride, calcium chloride, sodium lactateand the like. The concentration of active agent in these formulationscan vary widely, and will be selected primarily based on fluid volumes,viscosities, body weight and the like in accordance with the particularmode of administration selected and the patient's needs.

Thus, a typical pharmaceutical composition for intravenousadministration would be about 0.1 to 10 mg per patient per day. Dosagesfrom 0.1 up to about 100 mg per patient per day may be used,particularly when the drug is administered to a secluded site and notinto the blood stream, such as into a body cavity or into a lumen of anorgan. Substantially higher dosages are possible in topicaladministration. Actual methods for preparing parenterally administrablecompositions will be known or apparent to those skilled in the art andare described in more detail in such publications as Remington'sPharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa.(1980).

The pharmaceutical compositions can be administered in a variety of unitdosage forms depending upon the method of administration. For example,unit dosage forms suitable for oral administration include, but are notlimited to, powder, tablets, pills, capsules and lozenges. It isrecognized that the inhibitors of the invention (e.g. azumolene) whenadministered orally, should be protected from digestion. This istypically accomplished either by complexing the molecules with acomposition to render them resistant to acidic and enzymatic hydrolysis,or by packaging the molecules in an appropriately resistant carrier,such as a liposome or a protection barrier. Means of protecting agentsfrom digestion are well known in the art.

Further in accordance with the present invention, there are providedpharmaceutical preparations for administering to human or veterinarypatients, said preparations comprising RyR blockers such as above or apharmaceutically acceptable carriers, excipients and other ingredientscommonly used in pharmaceutical preparations for oral, rectal,intravenous, intraarterial, intradermal, subcutaneous, intramuscular,intrathecal, sublinginual, bucal, intranasal, trans-mucosal,trans-dermal, topical, other enteral, other parenteral and/or otherpossible route(s) of administration.

The compositions containing inhibitors of the invention can beadministered for therapeutic or prophylactic treatments. In therapeuticapplications, compositions are administered to a patient suffering froma disease (e.g., an autoimmune disease) in an amount sufficient to cureor at least partially arrest the disease and its complications. Anamount adequate to accomplish this is defined as a “therapeuticallyeffective dose.” Amounts effective for this use will depend upon theseverity of the disease and the general state of the patient's health.Single or multiple administrations of the compositions may beadministered depending on the dosage and frequency as required andtolerated by the patient. In any event, the composition should provide asufficient quantity of the agents of this invention to effectively treatthe patient. An amount of an inhibitor that is capable of preventing orslowing the development of a disease in a patient is referred to as a“prophylactically effective dose.” The particular dose required for aprophylactic treatment will depend upon the medical condition andhistory of the patient, the particular disease being prevented, as wellas other factors such as age, weight, gender, administration route,efficiency, etc. Such prophylactic treatments may be used, e.g., in apatient who has previously had a disease to prevent a recurrence, or ina patient who is suspected of having a significant likelihood ofdeveloping the disease.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Overexpression of RyR1 Gene Expression in Activated Human TCells Compared with Resting Human T Cells

Experimental Procedures

Cell Cultures and Chemicals—

All studies involving human subjects were performed after approval by UCDavis Institutional Review Board and after all subjects gave writteninformed consent. Resting CD3⁺ human lymphocytes were isolated from theperipheral blood of healthy consented adults using RosetteSep™ humanlymphocyte enrichment kit (Stem Cell Tech.) following the manufacturer'sinstructions. Resting T cells were resuspended at 0.5-1×10⁶ cells/ml inRPMI 164 medium supplemented with 10 mM HEPES, 10% fetal bovine serum,2% L-glutamine, 2% vitamins solution, 1% RPMI amino acids solution, and0.01% β-mercaptoethanol and maintained in suspension in 5% CO₂ at 37° C.Resting T cells were activated on the day of isolation by incubationwith either PHA (5-20 μM) or anti-CD3 antibody coated onto the tissueculture plates and soluble anti-CD28 antibody (5 μg/ml; BD Biosciences).Resting T cells were taken for analysis on the day of isolation, whereasactivated T cells were tested 4-5 days after activation.

Human acute T cell leukemia line, Jurkat E6-1 (ATCC, Manassas, Va.), wasmaintained in cell culture media containing RPMI 1640 medium(Lonza/BioWhittaker, Basel, Switzerland), 10% FBS (Omega Scientific,Tarzana, Calif.), 2% L-glutamine, 2% vitamins solution, 1% RPMI aminoacids solution, and 0.01% β-mercaptoethanol. Cells were kept insuspension in 5% CO₂ at 37° C. and passaged every 2 days.

Real-Time PCR—

Cells were lysed and relative quantifications of the expression levelsof human RyR1, RyR2, and RyR3 were performed using Real Time TaqMan PCRassays using AB Primer Express 2 software and commercially availableTaqMan human gene expression assay kits. The housekeeping gene B2M wasused for normalization. The human Real Time TaqMan® PCR assays used forB2M (Hs99999907m1), hRyR1 (Hs00166991m1), hRyR2 (Hs00892902m1), andhRyR3 (Hs01044126m1) were ordered directly from Applied Biosystems asinventoried gene expression assays. All assays were run in duplicates.

Data Analysis—

Statistical analysis was performed using Origin 7 software (OriginLab,Northampton, Mass.). All data are presented as the mean±standard errorof the mean; n=number of experiments. Statistical differences betweenmeans were accepted as statistically significant at p<0.05, usingStudent's independent t test.

Results

Resting T cells express at low level all three isoforms of RyR (RyR1,RyR2, and RyR3), whereas the expression of RyR1 and RyR2 issignificantly upregulated in activated T cells compared with resting (>5and >10 times, respectively; n=9) (FIG. 1 A, B). Interestingly, RyR3 wasexpressed at low level in resting T cells, whereas its expression wasnot detectable in activated T cell from 6 out of 9 donors tested. JurkatT cells express only RyR3 (not shown), which is consistent with aprevious reports (Guse A H, da Silva C P, Berg I, Skapenko A L, Weber K,Heyer P, Hohenegger M, Ashamu G A, Schulze-Koops H, Potter B V, Mayr GW. (1999) Nature (398) 70-73; Hakamata Y, Nishimura S, Nakai J,Nakashima Y, Kita T, Imoto K. (1999) FEBS Lett. (352) 206-210). Thus,these data indicate that activated human T cells overexpress theskeletal muscle-type hRyR1. Therefore, RyR1 inhibitors can be used toblock RyR1 in activated human T cells to reduce their pathogenicpotential in autoimmune diseases and graft vs. host disease. AlthoughRyR3 is expressed at low level in resting and activated T cells, theRyR3 potentially may be overexpressed in specific effector T cellsubsets and, therefore, inhibition of RyR3 potentially may haveimmunosuppressive effect.

Example 2 RyR Blockers Ryanodine and Dantrolene Suppress Cytosolic Ca²⁺Signaling in T Cells

Experimental Procedures

Calcium Imaging—

Cell cultures and chemicals were as described in the Example 1. Beforeexperiment, cells were plated on poly-l-lysine-coated glass-bottomclambers and then loaded with 1 μM fura-2/AM and pluronic F-127 for 5min in modified Tyrode solution containing (in mM): 130 NaCl, 5.6 KCl, 1MgCl₂, 2 CaCl₂, 10 HEPES, 10 D-glucose; pH 7.3. After washing, cellswere incubated for additional 30 min at 37° C. Some cells were incubatedin Modified Tyrode solution containing 400 μM Ry, 30 μM dantrolene, orvehicles (methanol or, ethanol, or DMSO) alone for 30 min at 37° C. Innominally Ca²⁺-free solution, Ca²⁺ was omitted.

Fluorescence images were acquired from adherent cells using a SenSys CCDcamera (Roper Scientific, Tucson, Ariz.) and a 40× oil immersion Zeissobjective on a Zeiss Axiovert 200 inverted microscope (Thornwood, N.Y.).Lambda DG-4 filter changer (Sutter Instrument, Novato, Calif.) was usedfor switching between 340 and 380 nm excitation wavelengths. Allexperiments were performed at room temperature. Solution exchange wasperformed via a gravity-driven perfusion system. Solution exchange inthe recording chamber was completed within 5 s. The vehicles-containingsolutions (methanol or DMSO) were applied in place of the drugs in allcontrol experiments. Data acquisition was performed using MetaFluor v7.0software (Universal Imaging, Downingtown, Pa.). [Ca²⁺]_(i) values wereestimated from fura-2 calibration as described previously (Grynkiewicz,G., Poenie, M., and Tsien, R. Y. (1985) J Biol Chem 260(6), 3440-3450).K_(d) was taken as 248 nM (Fanger, C. M., Neben, A. L., and Cahalan, M.D. (2000) J Immunol 164(3), 1153-1160). Unless otherwise indicated, ineach experiment changes in [Ca²⁺]_(i) were recorded from 15-20 cells andthen averaged.

Manganese Quench of Fura-2 Fluorescence—

Because Mn²⁺ readily passes through SOC channels (Missiaen, L.,Declerck, I., Droogmans, G., Plessers, L., De Smedt, H., Raeymaekers,L., and Casteels, R. (1990) J Physiol 427, 171-186) and is unlikely tobe transported from the cytosol (Chiesi, M., and Inesi, G. (1980)Biochemistry 19(13), 2912-2918; Chiesi, M., and Inesi, G. (1981) ArchBiochem Biophys 208(2), 586-592), the rate of fura-2 quenching in thepresence of Mn²⁺ was used as a measure of divalent cation influx intothe bulk cytoplasm in intact cells. The extracellular solution used inall Mn²⁺ quench experiments contained (in mM): 130 NaCl, 5.6 KCl, 1MgCl₂, 0.3 CaCl₂, 0.5 MnCl₂, 10 HEPES, 10 D-glucose; pH 7.3. Changes inCa²⁺-independent fluorescence due to Mn²⁺ quench were determined usingmethod described by Zhou & Neher (Zhou, Z., and Neher, E. (1993) JPhysiol 469, 245-273). Briefly, background corrected fura-2 fluorescenceexcited at 340 nm (F₃₄₀) was plotted against background corrected fura-2fluorescence excited at 380 nm (F₃₈₀) during the initial 10 secondsfollowing application of 30 μM cyclopiazonic acid (CPA). The slope ofthe F₃₄₀ versus F₃₈₀ relationship equals the ‘isocoefficient’ α. Thefluorescence value, F_(i), which is unaffected by changes in [Ca²⁺]_(i)was determined as: F_(i)=F₃₄₀+αF₃₈₀. The rate of F_(i) decline isproportional to the rate of Mn²⁺ entry into the cytosol and was used asan indicator of plasma membrane Ca²⁺ permeability.

Data Analysis—

Image analysis was performed using MetaFluor v7.0 software (UniversalImaging). Further measurements and statistical analysis were performedusing Origin 7 software (OriginLab, Northampton, Mass.). All data arepresented as the mean±standard error of the mean; n=number ofexperiments. Statistical differences between means were accepted asstatistically significant at p<0.05, using Student's paired or unpairedt tests.

Results

RyR Inhibitors Reduce Global [Ca²⁺]_(i) Elevation in Jurkat T Cells.

Fluorescent Ca²⁺ indicator fura-2, cyclopiazonic acid (CPA), areversible inhibitor of sarco-endoplasmic reticulum Ca²⁺-ATPase (SERCA),and ryanodine (Ry), an alkaloid which specifically blocks all types ofRyR (Gafni, J., Munsch, J. A., Lam, T. H., Catlin, M. C., Costa, L. G.,Molinski, T. F., and Pessah, I. N. (1997) Neuron 19(3), 723-733; Zucchi,R., and Ronca-Testoni, S. (1997) Pharmacol Rev 49(1), 1-51) wereemployed to explore the contribution of Ca²⁺ release from the RyR intothe global [Ca²⁺]_(i) dynamics in Jurkat T cells.

When CPA was applied in Ca²⁺-free solution, a transient elevation in[Ca²⁺]_(i) due to Ca²⁺ release via “leak” channels was observed (FIG.2A). Readdition of extracellular Ca²⁺ following first CPA applicationproduced a global elevation in [Ca²⁺]_(i) due to Ca²⁺ entry via SOCchannels. After removing of extracellular Ca²⁺, the second applicationof CPA evoked [Ca²⁺]_(i) transients, indicating that the store wasrefilled during extracellular Ca²⁺ readdition. The [Ca²⁺]_(i) transientsevoked by the first application of CPA were similar in shape andmagnitude in both untreated cells (control) and cells preincubated withRy (FIG. 2A). However, the [Ca²⁺]_(i), transients during Ca²⁺ readditionwere significantly smaller in Ry-treated cells compared withcontrol.-Furthermore, CPA application following Ca²⁺ readdition producedlarger [Ca²⁺]_(i) transients, in Ry-treated cells compared withuntreated cells.

Integration of [Ca²⁺]_(i) transients evoked by SERCA blockers has beenshown to provide an accurate estimate of releasable store content(Bergling, S., Dolmetsch, R., Lewis, R. S., and Keizer, J. (1998) CellCalcium 23(4), 251-259). The ratio of background-subtracted integrated[Ca²⁺]_(i) transients induced by the second CPA application (CPA₂) tothe first one (CPA₁) (FIG. 2A) was taken as measure of the extent ofstore refilling. The [Ca²⁺]_(i) transients recorded during Ca²⁺readdition induced by SOCE were also integrated and denoted as[Ca²⁺]_(SOCE). Summarized data presented in FIG. 2 show that applicationof Ry significantly downregulated [Ca²⁺]_(i) transients evoked by Ca²⁺readdition (FIG. 2 B) but facilitated store refilling (FIG. 2C) inJurkat T lymphocytes.

When applied extracellularly, Ry does not induce Ca²⁺ release (FIG. 2 D)or affect divalent ction influx (FIG. 2E), which confirmed thatinhibition of Ca²⁺ release from RyR accounts for the reduction in[Ca²⁺]_(i) elevation during Ca²⁺ readdition in cells preincubated withRy. Thus, activation of Ca²⁺ release from RyR contributes significantlyinto global [Ca²⁺]_(i) elevation in Jurkat T cells.

Inhibition of RyR Downregulates [Ca²⁺]_(i) Signaling Following TCRStimulation in Jurkat T Cells.

In the absence of extracellular Ca²⁺, a TCR agonist PHA evokes[Ca²⁺]_(i) elevation due to the Ca²⁺ release from the intracellularstore (FIG. 3 A, B). Pre-incubation with Ry did not significantly affectthe PHA- or CPA-induced [Ca²⁺]_(i) transients in the absence ofextracellular Ca²⁺ (FIG. 3 A-D), indicating that TCR crosslinking withPHA mobilizes Ca²⁺ from the IP₃- but not Ry-sensitive store.

When Ca²⁺ was readded following TCR stimulation to allow SOCE, theamplitudes of [Ca²⁺]_(i) transients were significantly lower inRy-pretreated cells than those in the untreated cells (FIG. 3 B, E). Onaverage, preincubation with Ry reduced the maximal amplitudes of[Ca²⁺]_(i) transients during Ca²⁺ readdition by 42% (FIG. 3 E),indicating that at physiological conditions Ca²⁺ release from RyR isactivated by extracellular Ca²⁺ influx and contributes significantly to[Ca²⁺]_(i) elevation.

Inhibition of RyR Downregulates [Ca²⁺]_(i) Signaling in Activated butnot Resting Human T Lymphocytes.

Consistent with upregulated expression of hRyR1 and hRyR2 genes inactivated human T cells, the RyR agonist caffeine produced Ca²⁺liberation from the ryanodine-sensitive store in the activated human Tcells but not in the resting (FIG. 1 C).

Stimulation with CPA in Ca²⁺-free extracellular solution evokedtransient elevation in [Ca²⁺]_(i) due to Ca²⁺ release from theintracellular store in both resting and activated human T cells (FIG. 4A, B). A second [Ca²⁺]_(i) transient was evoked by readdition ofextracellular Ca²⁺. The average values of integrated [Ca²⁺]_(i)transients evoked by Ca²⁺ readdition following store depletion were15.2±3.4 μM×sec (n=7) in resting T lymphocytes and 33.2±3.3 μM×sec (n=7)in activated T cells.

Furthermore, preincubation of the resting T cells with 30 μM dantrolene(Da), an inhibitor of RyR1 and RyR3 (Zhao, F., Li, P., Chen, S. R.,Louis, C. F., and Fruen, B. R. (2001) J Biol Chem 276(17), 13810-13816;Krause, T., Gerbershagen, M. U., Fiege, M., Weisshorn, R., and Wappler,F. (2004) Anaesthesia 59(4), 364-373), or blocking concentrations of Ry(500 μM) did not affect the time courses of changes in [Ca²⁺]_(i) (FIG.4A), indicating that contribution of Ry-sensitive Ca²⁺ store into global[Ca²⁺]_(i) signaling in resting T cells is not essential. In contrast,in activated T cells, both Ry and Da reduced [Ca²⁺]_(i) elevation evokedby readdition of extracellular Ca²⁺ (FIG. 4B, C). Thus, overexpressionof RyR1 mRNA in activated human T cells correlates with increasedcontribution of RyR-sensitive store into Ca²⁺ signaling in activated Tcells compared with resting, indicating that RyR1 play a significantrole in regulation of Ca²⁺ signaling in activated but not resting humanT cells and, therefore, may be used as a target for regulation ofCa²⁺-dependent functions of activated human T cells.

Example 3 RyR Blockers Ryanodine and Dantrolene Suppress Ca²⁺-DependentT Cell Functions

Experimental Procedures

Cell cultures and chemicals were as described in the Example 1.Proliferation assay—Cell division track assay was conducted usingcarboxyfluorescein succinimidyl ester (CFSE) proliferation kit(Molecular probes). Stock solution of CFSE (4 mM) was prepared in DMSO.Cells were washed, resuspended in PBS containing 4 μM CFSE at density1×10⁶ cells/ml, and incubated at 37° C. for 10 min. Labeling wasquenched by adding 5× volume of RPMI 1640 culture media containing 10%FBS. After washing 3 times with RPMI 1640+10% FBS, cells were placedinto FBS-free cell culture media to abolish cell division and keptovernight in 5% CO₂ at 37° C. After overnight incubation, theCFSE-labeled cells were pellet down and fraction of them (˜50000) werefixed with 1% paraformaldehyde in PBS and analyzed by flow cytometry toestablish the CFSE fluorescence profile of undivided cells. Theremaining CFSE-labeled cells were resuspended in cell culture mediasupplemented with 10% FBS at density 0.05×10⁶ cells/ml and seeded into24 well tissue culture plate (1 ml per well). Some cells werepre-incubated for 30 min at 37° C. with Ry (400 μM), or dantrolene (30μM) prior to transfer into FBS-containing cell culture media. Drugs werealso added to the same cells on each consecutive day of culturing. Thevehicles-containing solutions (methanol or DMSO) were applied in placeof the drugs in all control experiments. After 72 h in culture, cellswere harvested, washed with PBS, and fixed with 1% paraphormaldehyde inPBS. Fluorescence intensity was measured at 488 nm using FACScan flowcytometer and CellQuest software (Becton Dickinson Bioscience, MountainView, Calif.). The number of cell divisions was calculated using FlowJosoftware (Tree Star Inc, Ashland, Oreg.).

IL-2 Production Assay—

Jurkat T cells were washed 3 timed with PBS and resuspended in FBS-freecell culture media at density 0.8×10⁶ cells/ml and seeded into 96 welltissue culture plate (0.4 ml per well) and then stimulated with 100 μMmitogenic lectin phytohaemagglutinin P (PHA). Some cells werepre-incubated for 30 min at 37° C. with, Ry (400 μM), or dantrolene (30μM) prior to stimulation with PHA. After 24 hours of incubation, cellsculture supernatants were collected and contents of IL-2 was determinedby ELISA using Quantikine kits (R&D systems, Minneapolis, Minn.)according to manufacturer instructions.

Data Analysis—

Statistical analysis were performed using Origin 7 software (OriginLab,Northampton, Mass.). All data are presented as the mean±standard errorof the mean; n=number of experiments. Statistical differences betweenmeans were accepted as statistically significant at p<0.05, usingStudent's paired or unpaired t test or Wilcoxon nonparametric test.

Results

CFSE, a fluorescent dye that can be readily loaded into the cytoplasmand then equally distributed between cells after each cell division,resulting in a progressive decrease in CFSE fluorescence in cells thathave undergone multiple rounds of divisions (Wells, A. D.,Gudmundsdottir, H., and Turka, L. A. (1997) J Clin Invest 100(12),3173-3183). Because Jurkat T cells proliferate instantly withoutstimulation, the CFSE-labeled Jurkat T cells were first incubated inFBS-free medium to inhibit cell divisions. The proliferation was theninduced by transferring cells into complete cell culture media. Restinghuman CD3⁺ T cells were isolated from PBMC of healthy volunteers, thenlabeled with CFSE, and then were activated in vitro with anti-CD3/CD28Ab to induce proliferation.

Freshly isolated CD3⁺ resting human T cells or Jurkat T cells wereloaded with CFSE and fraction of them was fixed with 1% paraformaldehydein PBS and analyzed by flow cytometry to establish the CFSE fluorescenceprofile of undivided cells (time 0). The remaining CFSE-labeled cellswere resuspended in cell culture medium and activated with anti-CD3/CD28antibodies (human T cells) or transferred into FBS-containing meium(Jurkat T cells). Some cells were pre-incubated for 30 min at 37° C.with ryanodine (Ry, 400 μM) or dantrolene (Da, 30 μM) prior toactivation. Drugs were also added to the same cells on each consecutiveday of culturing. The vehicle-containing solutions (methanol or DMSO)were applied in place of the drugs to control cells. After 73 h (JurkatT cells) or 96 h (human T cells) of activation, cells were harvested,fixed, and CFSE fluorescence profiles were determined at 488 nm usingFACScan flow cytometer and CellQuest software (Becton DickinsonBioscience, Mountain View, Calif.). We found that RyR inhibitorssignificantly (p<0.05, Wilcoxon directional test, n=3 for eachcondition) reduced proliferation activity of Jurkat human T cells (FIG.5A). The peaks of CFSE fluorescence profiles obtained from the cellsexposed to RyR inhibitors were shifted to the right indicating slowercell cycle progression.

Preincubation with Ry, or Da significantly inhibited PHA-induced IL-2production (FIG. 5B), which was determined by ELISA. These data indicatethat RyR inhibitors are potent modulators of human T cell proliferationand TCR-mediated IL-2 production. The proposed mechanism of action ofRyR inhibitors on T cell functions is summarized in FIG. 6.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

What is claimed is:
 1. A method of treating a T-cell mediated skindisorder in a patient, the method comprising administering to thepatient a pharmaceutical composition comprising a ryanodine receptor(RyR) inhibitor and a pharmaceutically acceptable carrier, in the amountand form that is effective to treat or prevent the disease or disorder,wherein the RyR inhibitor is selected from the group consisting of anatural ryanoid, a synthetic ryanoid, and ryanodine.
 2. The method ofclaim 1, wherein the patient is a human.
 3. The method of claim 1,wherein the T-cell mediated skin disorder is an autoimmune skin disease.4. The method of claim 1, wherein the RyR inhibitor is administeredorally.
 5. The method of claim 1, wherein the RyR inhibitor isadministered parenterally.
 6. The method of claim 1, wherein the RyRinhibitor is administered topically.
 7. The method of claim 3, whereinthe autoimmune disease is psoriasis.
 8. The method of claim 1, whereinthe T-cell mediated disorder is allergic contact dermatitis.
 9. Themethod of claim 1, wherein the RyR inhibitor is ryanodine.